In recent years, there has been an increasing demand for development of a new alternative energy for reducing carbon dioxide all over the world due to the exhaustion of raw petroleum and increase in their prices which will occur in the near future, as well as the global warming caused by an increased amount of carbon dioxide emitted into the air. Bioenergy produced from biomass, which is one of earth resources that are reusable and rich among a variety of alternative energies, has come into the spotlight as a main renewable energy, which meets the above-described requirements. Particularly, bioethanol has been considered to be an alternative to a transportation fuel which is currently on high demand. For example, the use of bioethanol has already been obliged by the law in developed countries including the United States. Since raw materials currently used to produce bioethanol are limited to sugar, starch and the like derived from maize or other food resources (first-generation bio-fuel), they compete with mankind food resources, which results in an increase in price of international grains. As an alternative to solve these problems, a new land biomass (land biomass or lignocellulosic biomass??) (second-generation bio-fuel) or marine algal biomass (third-generation bio-fuel), which does not compete with the food resources, has also come into the spotlight as a next-generation bioenergy resource, and technologies for generating bio energy using the biomass has been on active research.
Since the marine (algal)biomass has a high content of polysaccharide usable by microorganisms, compared to the land (lignocellulosic)biomass, and is free from lignin, the marine biomass may be relatively easily pretreated, and harvested several times a year. In particular, since the Korean peninsula is surrounded by the sea on three sides except for the northern part, seaweeds may be used as a bio-resource, and their total annual yield is over 13,754 tons in 2006. In this aspect, the Republic of Korea belong to global seaweeds-producing countries including China, Japan and North Korea, but the usability of seaweeds other than edible resources is in a poor state (Korean fishery production statistics in 2006 by the Agriculture and Fisheries Production Statistics Division of the Population and Social Statistics Bureau in the Korea National Statistical Office).
Recently, much research on production of bioenergy from seaweeds has been conducted in Japan and Korea. In the name of the “Ocean Sunrise Project,” the Tokyo Fisheries Promotion Foundation has made a plane to produce five billion liters of fuel ethanol by farming a large amount of seaweeds in 4.47 million km2 of the exclusive economic zone and unused sea areas in the sea belt of Japan (Aizawa M et al., Seaweed bioethanol production in Japan—The Ocean Sunrise Project, OCEANS Conference, Sep. 29-Oct. 4, 2007, Vancouver, Canada). In Korea, a great interest has been focused in production of seaweeds since an ocean bio-fuel was included in the field of novel renewable energy, which is one of the 17 New Growth-driving Industries finally issued by the Korean Government on January of 2009. According to the presentation of development and research for technical exploration and utilization of ocean biomass issued by the Ministry for Food, Agriculture, Forestry and Fisheries in 2009, when seaweeds were cultured in a sea area of a regular square with 71 km each side to produce bioethanol, the bioethanol may be produced at an amount of 3.774 billion liters a year, which will replace 31.4% of an expected consumption of volatile oils in Korea in 2030.
Among the seaweeds recently known in the art, there has been active research on the use of red seaweed biomass (for example, Gelidium amansii) as a source material. Red seaweeds accounts for at least 70% of the total dry weight of polysaccharides, which may be converted into fermentable sugars that are usable by microorganisms. In particular, a main component of the polysaccharide derived from the red seaweed biomass is agar which accounts for approximately 60% of the total dry weight. Therefore, the red seaweed biomass has been considered to be a main resource for production of bioenergy. Agar polysaccharide is a polymer obtained by binding D-galactose and 3,6-anhydro-L-galactose (hereafter, abbreviated as “AHG”), which are used as monomer units, through α-1,3-linkage or β-1,4-linkage (Duckworth, M. and W. Yaphe (1971) Carbohydrate Research 16, 435-445) (see FIG. 1).
It was known that microorganisms using the agar polysaccharide as a carbon source use β-agarase or α-agarase to cleave the agar polysaccharide into smaller oligosaccharides. In this case, the oligosaccharides are finally degraded into α-neoagarobiose (or, α-1,3-D-galactosyl-3,6-anhydro-L-galactose) by the β-agarase, and finally degraded into β-agarobiose (or, β-1,4-anhydro-L-galactosyl-D-galactose) by the α-agarase. It was known that, when a degradation product of the β-agarase is neoagarobiose, the neoagarobiose should be converted into galactose so as to be metabolized by a microorganism, and thus α-neoagarobiose hydrolase cleaving an α-1,3 linkage is necessarily required for convention of the neoagarobiose into the galactose (Ekborg, N. A. et al (2005) Int. J. Syst. Evol. Microbiol. 55, 1545-1549; Ekborg, N. A. et al., (2006) Appl. Environ. Microbiol. 72, 3396-3405). However, an enzyme cleaving the α-1,3 linkage of neoagarobiose in S. degradas was not found until now (Ekborg, N. A. et al. (2006) Appl. Environ. Microbiol. 72, 3396-3405).
It was reported that β-agarase that produces oligoagarosaccharides from agarose in a microorganism is produced by many microorganisms such as, for example, a Pseudomonas sp. strain (Ha, J. C. et al. (1997) Biotechnol. Appl. Biochem. 26:1-6), an Alteromonas sp. strain (Potin, P., et al. (1993) Eur. J. Biochem. 214:599-607), an Agarivorans sp. strain (Ohta, Y. et al. (2005) Biotechnol. Appl. Biochem. 41:183-191), a Pseudoalteromonas sp. strain (Belas, R. (1989) J. Bacteriol. 171:602-605), a Microsilla sp. strain (Zhong, Z. et al. (2001) Appl. Environ. Microbiol. 67:5771-5779), and a Vibrio sp. strain (Aoki, T. et al. (1990) Eur. J. Biochem. 187:461-465).
When agar polysaccharide derived from red seaweed was used as a resource for production of bioenergy, the agar polysaccharide is necessarily converted into fermentable sugars, which can be actually used by a microorganism through a multiple pretreatment procedures. The conversion of the agar polysaccharide into fermentable monosaccharides may be performed through two processes: chemical pretreatment and biological pretreatment. First, a chemical method using acid hydrolysis is a relatively simple process, but a biomass composed of polysaccharides is chemically pretreated at a high temperature to mass-produce toxic by-products such as furfural and hydroxymethylfurfural (HMF), and to yield a mixture of randomly cleaved monosaccharide and oliogosaccharide (Pickering et al., 1993, Journal of Applied Phycology 5: 85-91; Armis'en, 1995). On the contrary, the biological pretreatment and saccharification methods using an enzyme such as β-agarase has an advantage in that they are environment-friendly methods performed at a room temperature to obtain a fermentable sugar such as galactose, but a currently commercially available enzyme is limited to β-agarase and a final product of the β-agarase is disaccharide (neoagarobiose or agarobiose) which may not used by conventional microorganisms.
Neoagarobiose produced from a reaction of the β-agarase should be necessarily converted into a fermentable monosaccharide such as galactose for use in production of bioenergy. In this case, α-neoagarobiose hydrolase is required. Therefore, in a final step of the effective biological (enzymatic) pretreatment and saccharification processes to use a red algal biomass as a resource for production of bioenergy such as bioethanol, α-neoagarobiose hydrolase is necessarily required. Also, AHG which is produced with galactose as degradation products of neoagarobiose was not commercially available, and may be purchased only as D-AHG, which is, however, very expensive (200 pounds (G.B.)/100 mg as of 2009, Dextra Laboratories). Therefore, it is possible to mass-produce an expensive, rare monosaccharide, AHG, from agarose using the enzyme of the present invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.